A major pathway by which human cells repair bulky DNA damage is the nucleotide excision repair pathway. The genetic and biochemical complexity of this process In humans has been revealed through the study of individuals with the cancer-prone genetic disease, xeroderma pigmentosum (XP). In XP cells, excision repair is partially to completely compromised, depending on which of at least 7 genes are affected. Recently two different genes have been cloned which, when individually introduced into XP-A cells, give rise to substantially enhanced levels of survival after UV irradiation challenge. One of these genes, that was identified in my laboratory, has been shown to restore the ability of XP-A cells to repair a variety of bulky lesions: cyclobutane dimers, anti-BPDE adducts and N-AcO-AAF lesions. However, In these same cells, restoration of (6-4) photoproduct repair proceeds at approximately 3% the rate that is measured in normal human cells. The objective of this research is to provide a basic understanding of the genetics and biochemistry of the mechanism(s) by which normal human cells repair bulky DNA modifications. Although our preliminary studies focused on observations concerning the phenotype of partially repair-proficient XP-A cells, a major component of this proposal focuses on the XP-A-correcting gene and its gene product. The complete XP-A-correcting gene will be identified from lamda/gt11 cDNA libraries, using the partial cDNA sequence that is already available. This complete cDNA wall be tailored for mammalian cell expression and reintroduced Into XP-A cells and those cells evaluated for enhanced resistance to a variety of DNA-damaging agents. With the gene sequence available, antibody reagents will be produced that will facilitate determining the role that this gene product plays in the initiation of repair and its interaction with other cellular proteins. Antibodies will also facilitate the purification of this protein either from human tissues or from a bacterial expression system. The purified protein will be used in vitro reconstruction assays and in biophysical characterization. In addition to these genetic and biochemical studies, a further understanding of this gene is warranted through cell biology studies. Specifically, new cell lines will be created in which both XP-A- complementing genes are introduced into the same cell-these cells should display full DNA-repair responsiveness. Also, preferential DNA repair studies will be performed that are designed to probe whether the overall repair process or some subset of repair (i.e., active gene ret)air) has been restored.